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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14641/m_MTBLS14641_LC-MS_negative_hilic_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14641/a_MTBLS14641_LC-MS_negative_hilic.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14641/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14641/s_MTBLS14641.txtprimary200OKftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14641Metabolites were identified using EL-MAVEN using retention time and accurate m/z measurements compared against an in-house library.MetaboLightsPublicLiquid Chromatography MS - negative - hilicLC-HRMS analysis was performed on a Vanquish UPLC and a Q-Exactive HF mass spectrometry, employing the same conditions as the previously established methods (79). A ZIC-pHILIC guard column (4.6 mm ID x 20 mm length, MilliporeSigma, Burlington, MA) and ZIC-pHILIC LC column (4.6 mm ID x 150mm length, 5 μm particle size, MilliporeSigma, Burlington, MA) were used for chromatographic separation at a column temperature of 30 °C. The sample injection volume was 5 μL. The mobile phases consisted of 10 mM (NH4)2CO3 and 0.05% NH4OH in H2O for mobile phase A, and 100% acetonitrile for mobile phase B. The LC gradient conditions were as follows: 0 to 13 min: 80% to 20% of mobile phase B, 13 to 15 min: 20% of mobile phase B. IFN-γ-driven iNOS induction in macrophages mediates CAR T cell resistance in B cell lymphoma (Glucose Tracer).John KoomenMoffitt Cancer CenterT cellmass spectrometry assayT cells were washed with 1 ml of ice-cold PBS, and metabolites were extracted with 300 μl of 80% methanol via incubation at -80 °C for 15 min. Samples were centrifuged (17,000 x g, 20 min, 4 °C), and supernatants were transferred to an Eppendorf tube and dried in a vacuum evaporator overnight. The dried extracts were resuspended in 20 μl of aqueous 50% methanol, clarified by centrifugation (17,000 x g, 20 min, room temperature), and analyzed by LC-HRMS.Mus musculushttps://www.ebi.ac.uk/metabolights/MTBLS14641Marco Davila. Roswell Park Comprehensive Cancer Center. 665 Elm St, Buffalo, NY 14203, USA. Marco.Davila@RoswellPark.org.John Koomen. Moffitt Cancer Center. 12902 Magnolia Drive Tampa, FL 33612 USA. john.koomen@moffitt.org.Raw data files were converted into cdf files using XCalibur file converter (Thermo). For metabolite isotope tracer experiments, the unlabeled or 13C-labeled metabolite peaks were extracted using EL-Maven with a metabolite standard-based in-house library. For the 13C-labeled metabolite peaks, the natural isotope peak area was corrected using IsoCor (Version 2.2). For data upload to MetaboLights, rawconverter.exe was used to create mzXML files from Thermo .raw data.L-NILCo-culturejohn.koomen@moffitt.orgFor 13C6-labeled glucose tracing of CAR T cells comparing the effects of no macrophages (NoMac) to unpolarized macrophages (unMac) and immunoregulatory macrophages (imMac) as well as L-NIL iNOS inhibitor treatment versus vehicle control, 1×10^6 T cells were resuspended in either RPMI-1640 medium (RPMI + 10% heat-inactivated dialyzed FBS) or 13C6-glucose substituted RPMI-1640 medium (glucose-free RPMI + 10% heat-inactivated dialyzed FBS + 11.1 mM 13C6-glucose). After 4 h incubation, cells were collected, rapidly centrifuged (17,000 x g, 10 sec, room temperature), and medium was removed. MetabolomicsMetabolomicsMus musculusuntargeted analysisT cellMacrophageschemical tracerexperimental sampleThermo Scientific Vanquish UHPLC SystemLC-MSCAR-TNitric Oxide SynthaseDiffuse large B cell lymphomaThermo Scientific Q Exactive HFMetabolomicsMus musculusuntargeted analysisT cellMacrophageschemical tracerexperimental sampleThermo Scientific Vanquish UHPLC SystemLC-MSCAR-TNitric Oxide SynthaseDiffuse large B cell lymphomaThermo Scientific Q Exactive HF LC-HRMS analysis was performed on a Vanquish UPLC and a Q-Exactive HF mass spectrometry, employing the same conditions as previously established methods. The ionization was set to negative mode, with the MS scan range set to 60 to 1000 m/z. The mass resolution was 70,000, and the AGC target was 1 x 106. falseIFN-γ-driven iNOS induction in macrophages mediates CAR T cell resistance in B cell lymphoma (Glucose Tracer)Chimeric antigen receptor (CAR) T cell therapies have revolutionized B cell malignancy treatment, but subsets of patients with large B cell lymphoma (LBCL) experience primary resistance or relapse after CAR T cell treatment. To uncover tumor microenvironment (TME)-induced resistance mechanisms, we examined patients’ intratumoral immune infiltrates and observed that elevated levels of immunoregulatory macrophages in pre-infusion tumor biopsies are correlated with poor clinical responses. In murine models, CAR T cell-produced interferon-gamma (IFN-g) promotes the expression of inducible nitric oxide synthase (iNOS, NOS2) in immunoregulatory macrophages, impairing CAR T cell function. Mechanistically, proteomics analysis of CAR T cells revealed that iNOS-expressing macrophages promote the upregulation of genes mediating apoptosis and cell cycle arrest in CAR T cells, while downregulating ribosome biogenesis and protein synthesis. Furthermore, CAR T cell metabolism is compromised by the depletion of glycolytic intermediates and rewiring of the TCA cycle. Pharmacological inhibition of iNOS enhances the CAR T cell treatment efficacy in B cell tumor-bearing mice. Notably, elevated levels of iNOS+CD14+ monocytes were observed in leukaphereses of patients with non-durable response to CAR T cell therapy. These findings suggest that mitigating iNOS in tumor-associated macrophages (TAMs), potentially by modulating IFN-gamma expression in CAR T cells, could improve outcomes for LBCL patients.2026-05-312026-05-31MTBLS14641